Method for the diagnosis of rheumatoid arthritis

ABSTRACT

A method for diagnosing rheumatoid arthritis, which is characterized by enzyme-immunologically measuring the amount of collagenase inhibitor present in sera, plasmas or synovial fluids by way of a sandwich assay wherein two different monoclonal antibodies which specifically bind to different antigenic determinants of the collagenase inhibitor are used, and comparing the measured amount with that for normal subjects.

FIELD OF THE INVENTION

The present invention relates to a method of the diagnosis of rheumatoid arthritis by way of quantitation of collagenase inhibitor. More particularly the invention relates to a method for carrying out the diagnosis of rheumatoid arthritis, by means of a sandwich enzyme immunoassay for human collagenase inhibitor using monoclonal antibodies to bovine collagenase inhibitor (tissue inhibitors of metallproteinases: TIMP), on the basis of the fact that the amount of collagenase inhibitor in sera, plasmas or synovial fluids from patients with rheumatoid arthritis is clearly higher than the amount of collagenase inhibitor in sera, plasmas or synovial fluids, respectively, from normal subjects. The enzyme immunoassay mentioned above is used to mean a method for determining collagenase inhibitor which is characterized in that there are used two different monoclonal antibodies which specifically bind to different antigenic determinants of collagenase inhibitor as an antibody to be bound to a solid phase support and an antibody to be labeled with an enzyme.

BACKGROUND ART

Among heretofore used methods for the diagnosis of rheumatoid arthritis may be mentioned: Rose's method based on the detection of rheumatoid factor, modified Rose's method by Heller, RAHA-test and RA-test. These methods, however, have such disadvantages that they use complex experimental methods or that it takes many days to go through with the diagnosis.

Collagenase inhibitor has been known to exist in human's and other animals' bones, skins, dental pulps, amniotic fluids, bloods and synovial fluids as well as in cultures of joint chondrocytes, synovial cells, fibroblasts derived from varied tissues and fibrosarcomatous cells. As means for determining the amount of collagenase inhibitor methods have heretofore been known which are based on measurement of its biological activity. As described by Eisen et al. in J. Lab. Clin. Med. 75, 258-263 (1973) and also by Cawston et al. in Arthritis and Rheumatism 27, 285-290 (1984), however, it is impossible, with the heretofore known methods of determination, to assay sera, plasmas or synovial fluids for collagenase inhibitor activity since there exist in such fluids proteins, such as α₂ -macroglobulin, which interfere disturbingly with the measurement. Hayakawa, Iwata et al. have already developed a sandwich enzyme immunoassay (EIA) using monoclonal antibodies to bovine collagenase inhibitor, a method which enables specific quantitation of collagenase inhibitor with very small volumes of samples in a precise, straightforward and rapid manner (Japanese Patent Application No. Sho 62-42781). The present inventors have discovered that the level of collagenase inhibitor present in human sera, plasmas or synovial fluids apparently increases in association with affliction with rheumatoid arthritis, and found that it is possible to make a diagnosis of rheumatoid arthritis by determining, by the enzyme immunoassay mentioned above, the amount of collagenase inhibitor present in sera, plasmas or synovial fluids.

DISCLOSURE OF THE INVENTION

The present invention provides a method for diagnosing whether the subject is afflicted with rheumatoid arthritis, which comprises carrying out an enzyme immunoassay, with the aid of monoclonal antibodies that specifically bind to different antigenic determinants of bovine collagenase inhibitor as antibodies to be bound to a solid phase support and to be labeled with an enzyme, to measure the amount of human collagenase inhibitor present in the subject's sera, plasmas or synovial fluids, and comparing the measured amount with that for normal subjects.

In the above mentioned enzyme immunoassay employed according to the method of the present invention, a variety of materials in any usage forms which well adsorb passively antigens or antibodies may be chosen for use as solid supports, e.g. polystyrenes, polycarbonates, polypropylenes or polyvinyls in such forms as balls, microplates, sticks or test tubes. As the antibody to be labeled with an enzyme, there may be used an IgG fraction obtainable by the fractionation of a material containing antibodies with ammonium sulfate followed by purification on an anion exchange gel such as DEAE-Sephacel, as well as Fab' that specifically reacts with an antigen molecule and that is obtainable by its digestion with pepsin followed by reduction.

The method of the invention is for diagnosing whether the subject is afflicted with rheumatoid arthritis by carrying out the quantitative determination of human collagenase inhibitor on the basis of an enzyme immunoassay with the aid of a combination of two monoclonal antibodies that specifically bind to different antigenic determinants of the collagenase inhibitor as antibodies to be bound to a solid phase support and to be labeled with an enzyme, followed by comparing the result with values for normal subjects.

The present invention will now be illustrated in more detail by way of the following examples, but it is to be noted that this invention is not limited thereto.

EXAMPLE 1 Preparation of Monoclonal Antibodies Against Bovine Collagenase Inhibitor

(a) Preparation of Antigen-Bovine Collagenase Inhibitor

In accordance with the procedure described by the present inventors in J. Biochem. 96, 395-404 (1984), collagenase inhibitor was purified obtained by cultivated root pulps from bovine unerupted wisdom teeth in Eagle's MEM medium (supplied by Nissui Seiyaku) using columns of Con A-Sephorose, Ultrogel AcA 44 and DE-52 cellulose. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method described by Laemmli et al. in J. Mol. Biol. 80, 579-599 (1973), the purified inhibitor showed a single band having a molecular weight of about 32,000 dalton (D).

(b) Preparation of Antibody-Producing Cells

Two 6-week-old female Balb/c mice were initially immunized intraperitoneally with 48 μg in 0.4 ml emulsion of purified bovine collagenase inhibitor described above in (a) in Freund's complete adjuvant. The mice then received, after 30 days, a booster injection of 84 μg of the bovine collagenase inhibitor dissolved in saline. As the final immunization, the mice received, after 58 days, an additional booster injection by intraperitoneal administration of 95 μg/500 μl of the inhibitor in saline. On the 3rd day after the final immunization the spleen was extirpated and the spleen cells were prepared.

(c) Cell Fusion

(1) Materials and methods used are as follows:

RPMI 1640 culture medium: RPMI No. 1640 (Difco Laboratories) supplemented with sodium bicarbonate (12 mM), sodium pyruvate (1 mM), L-glutamine (2 mM), penicillin G potassium (50 U/ml), streptomycin sulfate (50 μg/ml) and amikacin sulfate (100 μg/ml), adjusted at pH 7.2 with dry ice and filtered through a 0.2 μm Toyo membrane filter for sterilization.

NS-1 culture medium: the above RPMI 1640 culture medium supplemented with 15% (v/v) fetal bovine serum (M.A. Bioproducts) filter-sterilized.

PEG 4,000 solution: Serum-free RPMI 1640 culture medium, containing 50% (w/w) polyethylene glycol 4,000 (PEG 4,000, Mercc & Co., Inc.).

Utilizing 8-azaguanine-resistant myeloma cell line, NS-1 (P3-NS1-1), cell fusion was effected according to a slightly modified method of Oi et al. described in "Selected Method in Cellular Immunology" (ed. B. B. Mishell and S. M. Shiigi, W. H. Freeman and Company, (1980), pp. 351-372).

(2) The karyo-splenocytes prepared as described in the preceding (b) (cell viability 100%) were fused in a ratio of 5 to 1 with myeloma cells (cell viability 100%). Thus, the splenocytes and the myeloma cells were separately washed in RPMI 1640 culture medium, then suspended in the same medium and mixed together for fusion in the ratio described above. 40 ml of the RPMI 1640 culture medium was put into a 50 ml conical styrene resin test tube (Iwaki Glass), and centrifuged at 400 xg for 10 min., and the supernatant was discarded completely by suction. To the precipitated cells was added dropwise over 1 min. 1.3 ml of the PEG 4,000 solution warmed at 37° C. with gentle stirring. The cells were resuspended and dispersed by stirring for another minute. To the suspension was then added dropwise over 1 min. 1.3 ml of RPMI 1640 culture medium warmed at 37° C. After repeating the same operation once more, the cells were dispersed, by adding 9 ml of the same culture medium dropwise over 2 to 3 min. under continuous agitation. The dispersion was centrifuged at 400 xg for 10 min., and the supernatant was discarded completely by suction. To the precipitated cells was added rapidly 12.9 ml of NS-1 culture medium warmed at 37° C., and big cell clumps were then dispersed by pipetting carefully with a 10 ml pipette. The dispersion was diluted with 26 ml of the same culture medium, and distributed into 96-well microwell made of polystyrene (Iwaki Glass) so that 6.0×10⁵ cells/0.1 ml exist in each well. As a preliminary treatment, the 96-well microwells had been treated with 0.2 ml of NS-1 culture medium overnight at 37° C. in a CO₂ incubator, and the culture medium had been removed therefrom by suction on actual use. After loaded with the cells, the microwells were subjected to cultivation at 37° C. in 7% CO₂ /93% air under 100% humidity.

(d) Selective Proliferation of Hybridomas with the Aid of a Selective Culture Medium

(1) Culture media used are as follows

HAT selection medium: the NS-1 culture medium mentioned above in (c) supplemented with hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine (16 μM).

HT culture medium: the same composition as the HAT medium mentioned above except in that aminopterin is not contained.

(2) One day after the cultivation mentioned above in (c) was started (i.e. on the first day), two drops (ca. 0.1 ml) of HAT culture medium were added with a Pasteur pipette. On the 2nd, 3rd, 5th, 8th and 11th days, the half (0.1 ml) of each culture medium was replaced with fresh HAT culture medium. On 14th day, the half of each culture medium was replaced with fresh HT culture medium. Thereafter, replacement of the half of each culture medium by fresh HT culture medium was repeated at 3 to 4 days intervals. Usually, sufficient growth of hybridoma was observed in 2 to 3 weeks. All the wells containing hybridomas grown were tested to see if there are positive wells, using a solid phase-antibody binding test (ELISA) as will be described in the subsequent (e). All the cultures with hybridomas positive in the test above were transferred to a 24-well cell well made of polystyrene (Iwaki Glass) containing 1 ml of the HT culture medium containing 10⁷ mouse thymocytes as feeders. The cultures were subjected to cultivation, as described in the preceding (c), at 37° C. in 7% CO₂ for about a week. Once or twice during the cultivation period 0.5 ml of the supernatant of each well was replaced with 0.5 ml of fresh HT culture medium. At the time the sufficient growth of hybridomas was observed, the cultures were checked again for the positivity by ELISA method, and subjected to a cloning procedure by the limiting dilution method as will be explained in (f) below. The residual culture after cloning was transferred into a 25 cm² tissue culture flask made of polystyrene (Iwaki Glass), for the preparation of a frozen sample for storage.

(e) Screening for hybridomas capable of producing anti-bovine collagenase inhibitor antibody by means of the solid phase-antibody binding test (ELISA):

A method used for the present example was a slight modification of the method of Rennard et al. described in Anal. Biochem., 104, 205-214, (1980) which is appropriate for the detection of antibody production in hybridoma. Each well of a 96-well microtitration plate (Flow Laboratories, Inc.) was coated with 0.5-1.0 μg of bovine collagenase inhibitor and the wells remained uncoated were blocked with 1% bovine serum albumin (BSA). To the wells was added a part of the supernatant from the wells containing hybridomas grown as obtained in (d) above. The incubation was carried out at room temperature for about 1 hr. After the addition of goat anti-mouse immunoglobulin (Cappel Lab.) labeled with horseradish peroxidase as a secondary antibody, further incubation was carried out at room temperature for about 1 hr. The depth of brown color produced upon the addition of H₂ O₂ and o-phenylenediamine as a substrate was assessed qualitatively with naked eyes or determined by measuring the absorbance at 500 nm using a Corona double wave microplate spectrophotometer (MTP-22, Corona Electric).

(f) Cloning

In order to obtain hybridomas capable of producing monoclonal antibodies it is necessary, after the procedure mentioned above in (d), for cloning to be carried out according to the limiting dilution method, since each well can develop two or more kinds of hybridomas. A cloning culture medium containing in NS-1 culture medium 10⁷ cells per ml of mouse thymocytes as feeders was prepared. 5, 1 and 0.5/well of hybridomas were added to 36, 36 and 24 wells of a 96-well microwell, respectively. About 0.1 ml of NS-1 culture medium was further added to each well on the 5th and 12th day. About 14 to 15 days after the cloning when the sufficient growth of hybridoma could be recognized, the culture group in which more than 50% wells were negative for colony formation was subjected to ELISA. When not all of the tested wells were shown to be positive, the colonies in antibody-positive wells were enumerated and out of them 4-6 wells exhibiting the formation of one and single colony were selected for recloning. Finally, 17 hybridoma clones capable of producing monoclonal antibody to bovine collagenase inhibitor were obtained.

(g) Proliferation in vitro and in vivo of Monoclonal Antibody

Monoclonal antibodies were proliferated in a conventional manner. Thus, they can be obtained from the supernatant of an appropriate culture medium such as NS-1 culture medium in which each of the resultant hybridomas were subjected to cultivation (in vitro proliferation) (The concentration of the monoclonal antibody was 10-100 μg/ml). If a large amount of antibody is to be produced, on the other hand, an in vivo proliferation is carried out as described below. A carcinogenic excitant, Pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chemical), was administered to animals (Balb/c mice) intraperitoneally in a volume of 0.5 ml per animal, which animals were of the syngenic strain as those providing the splenocytes and the myeloma cells. One to 3 weeks after the administration, 1×10⁷ cells of each hybridoma were administered intraperitoneally as above, and ascites containing 4-7 mg/ml of monoclonal antibody were obtained after 1 to 2 weeks.

(h) Isotypes of Monoclonal Antibodies

First of all, each of the ascites obtained in (g) above was bound to a microtitration plate coated with bovine collagenase inhibitor according to the above-described ELISA method. After washing with PBS, isotype-specific rabbit anti-mouse Ig antibodies (Zymed Laboratories) were added thereto. After washing with PBS, goat anti-rabbit IgG (H+L) antibody labeled with horseradish peroxidase, was added thereto, and 2,2'-azinodi(3-ethylbenzothiazoline sulfate-6) as a substrate and hydrogen peroxide were then added for detection. The results are shown collectively in Table 1 described hereinafter. Among the resultant monoclonal antibodies against bovine collagenase inhibitor, fifteen antibodies were found to contain immunoglobulin chain γ1/κ and one antibody γ2a/κ and one antibody γ2b/κ.

(i) Purification of Monoclonal Antibody

Each of the ascites obtained in the foregoing (g) was fractionated with ammonium sulfate (40% saturation) and then loaded on DEAE Sephacel (Pharmacia) equilibrated with a 40-mM phosphate buffer solution (pH 8.0) containing 0.06-M NaCl to separate an unadsorbed fraction. This IgG fraction was gel-filtered on Sephacryl S-300 Superfine (Pharmacia) column equilibrated with a 50-mM phosphate buffer solution (pH 7.4) containing 0.42-M NaCl, whereby FCS from culture medium and mouse-derived proteins were separated and removed.

EXAMPLE 2 Reactivity Between Bovine Dental Pulp Collagenase Inhibitor and Monoclonal Antibodies

(a) Preparation of Enzyme-Labeled Monoclonal Antibodies (Fab'-POD Conjugates)

(1) Preparation of Fab' fraction

The IgG fraction obtained in Example 1(i) was dissolved in a 0.1-M acetate buffer solution, pH 4.2, containing 0.1-M NaCl and the solution was digested with pepsin as follows. Thus, pepsin was added to the fraction to 2% (w/w) based on the IgG therein, and the mixture was subjected to digestion at 37° C. for 24 hrs. The digested product was adjusted to a pH of 7.0 with a 2-M Tris solution thereby to stop the digestion reaction. The product was gel-filtered on a Ultrogel AcA 44 column (supplied by LKB) equilibrated with a 0.1-M phosphate buffer solution (pH 7.0) to obtain F(ab')₂ fraction.

The F(ab')₂ fraction was dialyzed against a 0.1-M phosphate buffer solution (pH 6.0) containing ethylenediamine tetraacetic acid (EDTA), and aminoethanethiol (MEA) was added to a final concentration of 10-mM to effect reduction at 37° C. for 1.5 hrs. The product was gel-filtered on a Ultrogel AcA 44 column equilibrated with a 0.1-M phosphate buffer solution (pH 6.0) containing 5-mM EDTA to obtain Fab' fraction.

(2) Preparation of Maleimide-Labeled POD Fraction

Separately from the procedure as mentioned in (1) above, horseradish peroxidase (POD) was labeled with a maleimide compound as follows. Thus, the POD was dissolved in a 0.1-M phosphate buffer solution (pH 7.0) to 10 mg/ml and to the solution was added 25 moles, per mole of the POD, of N-(ε-maleimidocaproyloxy)succinimide (EMCS) as a solution in dimethylformamide for reaction at 30° C. for 30 min. The product was gel-filtered on a Sephadex G-50 column equilibrated with a 0.1-M phosphate buffer solution (pH 6.0) to obtain maleimide-labeled POD fraction.

(3) Preparation of Fab'-POD Conjugate Fraction

The fractions as prepared in (1) and (2) above were admixed so that equimolar amounts of Fab' and maleimide-labeled POD in the respective fractions were mixed. The mixture was then diluted with a 0.1-M phosphate buffer solution (pH 6.0) containing 5-mM EDTA to a final concentration, in respect of both Fab' and maleimide-labeled POD, of 100-μM. After reaction at 4° C. for 20 hrs. was added 10 moles, per mole of Fab', of N-ethymaleimide to block unreacted thiol groups. The product was gel-filtered on a Ultrogel AcA 44 column equilibrated with a 0.1-M phosphate buffer solution (pH 6.5) to obtain Fab'-POD conjugate fraction, to which 0.1% bovine serum albumin (BSA) and 0.005% thimerosal were added for storage at 4° C.

(b) Western Blotting

The bovine dental pulp collagenase inhibitor purified in Example 1(a) was subjected to SDS-PAGE and then to Western blotting, in accordance with the method of Tanabe as described in Saibo Kogaku (Cell Technology) 1 and 2, pp. 1061-1068 (1983), using commercially available POD-labeled goat anti-mouse immunoglobulin and the Fab'-POD conjugates obtained in Example 2(a) above, whereby patterns of enzyme antibody staining were obtained. These patterns are shown in FIG. 1. In FIG. 1, A and B show the results obtained when a nitrocellulose membrane was, subsequently to the Western blotting, immunologically stained with Fab' (clone 7-3F1)-POD and Fab' (clone 7-21B12)-POD conjugates obtained in Example 2(a), respectively.

Futhermore, numbers 1 through 16 show the results obtained when a nitrocellulose membrane was, subsequently to the Western blotting, soaked in each of the following monoclonal antibodies (all of IgG type) in solution and then immunologically stained again with POD-labeled goat anti-mouse immunoglobulin (supplied by Cappel Laboratories):

    ______________________________________                                          1: clone 7-3F1      2: clone 7-4F2                                             3: clone 7-5A1      4: clone 7-6C1                                             5: clone 7-7F11     6: clone 7-8B2                                             7: clone 7-9B4      8: clone 7-10E11                                           9: clone 7-11A5    10: clone 7-12B6                                           11: clone 7-15E8    12: clone 7-18F3                                           13: clone 7-19F6    14: clone 7-20C2                                           15: clone 7-21B12   16: clone 7-23G9.                                          ______________________________________                                    

As is evident from FIG. 1, all the monoclonal antibodies mentioned above were found to have reactivity with bovine dental pulp collagenase inhibitor.

EXAMPLE 3 Sandwich Enzyme Immunoassay

(a) Preparation of Monoclonal Antibody-Coated Balls

According to the method of Ishikawa et al. as described in J. Immunoassay 4, 209-327 (1983), monoclonal antibody obtained in Example 1(i) was dissolved in a 0.1-M phosphate buffer solution (pH 7.5) containing 0.1% sodium azide. The monoclonal antibody solution was adjusted to a concentration of 100 μg/ml (A₂₈₀ =0.15) and polystyrene balls (6.5 mm in diameter, supplied by Precision Plastic Ball) were soaked therein and allowed to stand at 4° C. for 24 hrs. After removal of the monoclonal antibody solution, the balls were washed five times with a 10-mM phosphate buffer solution (pH 7.0) containing 0.1% BSA, 0.1M NaCl and 0.1% sodium azide (hereinafter referred to Buffer A for short), and then soaked in Buffer A for storage at 4° C.

(b) Sandwich Assay

A solution of purified collagenase inhibitor or a sample solution containing collagenase inhibitor was diluted with Buffer A containing 1% BSA and a 300 μl aliquot of dilution was placed in each test tube. After addition of an antibody-coated ball prepared in (a) above, the test tube was warmed at 37° C. for 1 hr. with shaking (first reaction) and then washed three times with 3 ml portions each of a 10-mM phosphate buffer solution (pH 7.0) containing 0.1-M NaCl. The Fab'-POD conjugate prepared in Example 2(a) was diluted to 20 ng/tube with a 10-mM phosphate buffer solution (pH 7.0) containing 0.1% BSA and 0.1-M NaCl, and then warmed at 30° C. for 1 hr. with shaking (second reaction). After the reaction was complete, washing was carried out in the same manner as upon the completion of the first reaction. 0.3 ml of a substrate for POD dissolved in a 0.1-M acetate buffer solution (pH 5.5), i.e. 0.0134% tetramethylbenzidine (TMBZ) was added. After further addition of 0.1 ml of 0.01% hydrogen peroxide, the mixture was warmed at 30° C. for 1 hr. with shaking (third reaction), whereafter the reaction was stopped by the addition of 0.6 ml of 1.33N sulfuric acid. The A₄₅₀ value of the reaction mixture was measured on a spectrophotometer and the amount of collagenase inhibitor in the sample was determined from a standard curve.

(c) Screening for Monoclonal Antibodies for Use in Sandwich Assay

In order to screen for monoclonal antibody combinations with which the quantitation of collagenase inhibitor is possible, Fab'-POD conjugate was prepared from each of the following monoclonal antibodies purified by the method as used in Example 1(i): clone 7-3F1; 7-6C1; 7-19F6 and 7-21B12. Using 1 ng/tube of purified bovine dental pulp collagenase inhibitor was carried out a quantitative sandwich assay, according to the method as used in Example 3(b), with each of the following monoclonal antibodies as solid phase produced from the following cell lines: clone 7-3F1; 7-4F2; 7-5A1; 7-6C1; 7-7F11, 7-8B2; 7-9B4; 7-10E11; 7-11A5; 7-12B6; 7-15E8; 7-18F3; 7-19F6; 7-20C2; 7-21B12 and 7-23G9. The cloned cell lines 7-6 C1 and 7-23G9 were deposited at the Fermentation Research Institute Agency of Industrial Science and Technology under the terms of the Budapest Treaty on Apr. 17, 1991 under the accession numbers FERM BP-3468 and FERM BP-3469, respectively. The resultant A₄₅₀ values are shown in Table 2 below. It is to be noted the A₄₅₀ values in Table 2 are those obtained by subtracting values for sandwich assay in which no collagenase inhibitor was added from those for sandwich assay in which 1 ng of sample was added. All the four different Fab'-POD conjugates mentioned above were found to show A₄₅₀ values not smaller than 2 for any of the six different antibodies as solid phase: 7-4F2; 7-11A5; 7-12B6; 7-18F3; 7-20C2 and 7-23G9. A quantitative sandwich assay was further carried out by using different amounts of bovine dental pulp collagenase inhibitor added for each of these twenty four combinations. The results obtained when Fab' (clone 7-6C1)-POD conjugate was used in combination with clone 7-23G9 antibody as the solid phase are shown in FIG. 2. As is shown by FIG. 2, linearity was obtained between the amount of bovine dental pulp collagenase inhibitor added and A₄₅₀, the determination sensitivities being about 1 pg (32 a mol) per tube. Such linearity was also observed with combinations other than the one mentioned above, it being thereby established that quantitative sandwich assay is possible with any of the combinations.

EXAMPLE 4 Identification of Collagenase Inhibitor in Human Serum

(a) Preparation of Affinity Column

In accordance with the method as described by Axen et al. in Nature 214, 1302-1304 (1967) and by Cuatrecasas et al. in Proc. Natl. Acad. Sci. USA, 61, 636-643 (1968), purified monoclonal antibody obtained in Example 1(i) was immobilized as ligand onto a Sepharose 4B support by way of the use of cyanogen bromide. The resultant antibody-Sepharose 4B gel (0.3 ml) was then filled into a glass column and equilibrated, before use, with a 30-mM Tris-HCl buffer solution (pH 8.0) containing 0.1-M NaCl and 5-mM CaCl₂.

(b) Affinity Chromatography for Human Serum Collagenase Inhibitor

Human serum (1 ml) was dialyzed against a 30 mM Tris-HCl buffer solution (pH 8.0) containing 0.1-M NaCl and 5-mM CaCl₂ and then applied onto a clone 7-21B12 antibody-Sepharose 4B column as prepared in accordance with the manner mentioned in (a) above. The column was washed with the same buffer as above to obtain "non-adsorbing fraction". It was then washed with a 30-mM Tris-HCl buffer solution (pH 7.5) containing 2-M NaCl and subsequently with a 0.2-M glycine-NaOH buffer solution (pH 10.5) containing 0.5-M NaCl to obtain "washed fraction". Protein adsorbed on the column was finally eluted with a 0.2-M glycine-HCl buffer solution (pH 2.0) to obtain "eluted fraction". The thus obtained eluted fraction was dialyzed against a 30-mM Tris-HCl buffer solution (pH 8.0) containing 0.1-M NaCl and 5-mM CaCl₂, and then subjected again to affinity chromatography on a clone 7-21B12 antibody-Sepharose 4B column to repeat the same procedure as described above, whereby collagenase inhibitor was obtained in the eluted fraction. In order to test whether the monoclonal antibodies against bovine dental pulp collagenase inhibitor react with human serum collagenase inhibitor, the eluted fraction mentioned above was subjected to SDS-PAGE and then to Western blotting. FIG. 3 shows the results obtained when a nitrocellulose membrane was, subsequently to the Western blotting, immunologically stained with Fab'-POD conjugate prepared from each of the following monoclonal antibodies: 1, clone 7-3F1; 2, clone 7-6C1; 3, clone 7-19F6; 4, clone 7-21B12; and 5, clone 7-23G9. As is shown in FIG. 3, collagenase inhibitors with reactivity with monoclonal antibody against bovine dental pulp collagenase inhibitor were found to exist in human serum. On top of that, it has also been found that the molecular weight of each of these collagenase inhibitors was the same as that (32,000D) of bovine dental pulp collagenase inhibitor obtained in Example 1(a).

EXAMPLE 5 Quantitative Determination of Collagenase Inhibitor in Human Serum by Sandwich Assay

(a) Preparation of Standard Curve

Screening was performed for the most suitable monoclonal antibody combination for conducting sandwich assay for human collagenase inhibitor. Thus, as shown in Example 3(c), twenty four combinations between four different Fab'-POD conjugates, i.e. Fab'(7-3F1)-POD, Fab'(7-6C1)-POD, Fab'(7-19F6)-POD and Fab'(7-21B12)-POD, and 6 different monoclonal antibodies as solid phase antibodies, i.e. clones 7-4F2, 7-11A5, 7-12B6, 7-18F3, 7-20C2 and 7-23G9, were screened for those with which quantitative determination of human serum collagenase inhibitors treated by column chromatography in the manner as described in Example 4(b) is possible. As a result, when human serum collagenase inhibitor was used as antigen, no A₄₅₀ signals were detected for most of the combinations used. It has been found, however, that quantitative sandwich assay is possible with the combination of clone 7-23G9 antibody as the solid phase antibody and Fab'(clone 7-6C1)-POD as the conjugate. A standard curve was prepared by performing the quantitative sandwich assay in accordance with the method as described in Example 3(c) with varied amounts of human serum collagenase inhibitor added, using the combination mentioned above. The results thus obtained are shown in FIG. 4. As is shown by FIG. 4, linearity is attained between the amount of human serum collagenase inhibitor added and A₄₅₀, it being thereby found that quantitative determination of human collagenase inhibitor is possible. The determination sensitivity, however, was found to be about 10 pg (320 a mol) per tube, ten times lower than that (i.e. 1 pg per tube) for bovine dental pulp collagenase inhibitor.

(b) Quantitative Determination By Sandwich Assay Of Collagenase Inhibitor In Sera Of Normal Subjects And Of Patients With Rheumatoid Arthritis

Using the monoclonal antibody combination specified in (a) above, fifty serum samples from normal subjects and fourteen from patients with rheumatoid arthritis were sandwich-assayed quantitatively for collagenase inhibitor contained therein. The results are shown in Table 3 below. In this sandwich assay, there was used the human serum collagenase inhibitor purified in Example 4(b) as the antigen for the preparation of the standard curve. This quantitation was performed with the serum samples diluted 1,600 times with Buffer A containing 1% BSA. The values indicated in Table 3 are expressed as means of duplicate experiments. As is seen from Table 3, the mean amount of collagenase inhibitor present in one ml of serum from patients with rheumatoid arthritis was as high as 2.08±0.58 μg (p<<0.001), whereas that from normal subjects was 1.23±0.20 μg. Thus, it is possible, by quantitating collagenase inhibitor in serum by the sandwich assay, to know whether the subject is afflicted with rheumatoid arthritis.

(c) Quantitative Determination By Sandwich Assay Of Collagenase Inhibitor In Synovial Fluids From Normal Subjects And From Patients With Rheumatoid Arthritis

Using the same method as shown in (b) above, seven samples of synovial fluid from normal subjects and five such samples from patients with rheumatoid arthritis were sandwich-assayed quantitatively for collagenase inhibitor contained therein. The results are shown in Table 4 below. As is seen from Table 4, the mean amount of collagenase inhibitor present in one ml of synovial fluid from patients with rheumatoid arthritis was as high as 9.49±1.77 μg (P<<0.001), whereas that from normal subjects was 2.35±0.36 μg.

Thus, it is possible, by quantitating collagenase inhibitor in synovial fluid by the sandwich assay, to know whether the subject is afflicted with rheumatoid arthritis.

EXAMPLE 6 Purification of Human Serum Collagenase Inhibitor

As mentioned above, in the eluted fraction finally obtained in Example 4(b) is present collagenase inhibitor with reactivity with monoclonal antibodies against bovine dental pulp collagenase inhibitor (see FIG. 3). A variety of proteins, however, were observed to exist therein, in addition to the collagen inhibitor, which do not react with the monoclonal antibodies mentioned above. Based on this observation, the eluted fraction was gel-filtered on an AcA 44 column equilibrated with a 0.1-M phosphate buffer solution (pH 7.5) for separation of collagenase inhibitor from other proteins thereby to purify human serum collagenase inhibitor. FIG. 5 shows the respective SDS-PAGE patterns for the resultant serum collagenase inhibitor (1), bovine dental pulp collagenase inhibitor (2) as a control and molecular weight markers (3). As is seen in FIG. 5, the molecular weight of human serum collagenase inhibitor was found to be the same as that (i.e. 32,000D) of bovine dental pulp collagenase inhibitor.

EXAMPLE 7 Preparation of Standard Curves by Sandwich Assay for Purified Human Serum Collagenase Inhibitor

A standard curve was prepared by performing the sandwich assay quantitatively with varied amounts of the purified human serum collagenase inhibitor obtained in Example 6 added, using the monoclonal antibody combination as mentioned in Example 5(a). The results thus obtained are shown in FIG. 6. As is seen from FIG. 6, linearity is attained between the amount of human serum collagenase inhibitor added and A₄₅₀, the determination sensitivity being 1.5 pg (48 a mole) per tube. This determination sensitivity was found to be 6.7 times higher than in the case of the standard curve prepared in Example 5(a) and 1.5 times lower than that for bovine dental pulp collagenase inhibitor as described in Example 3(c).

EXAMPLE 8 Diagnosis of Rheumatoid Arthritis by Way of Quantitation of Collagenase Inhibitor in Human Body Fluids by Sandwich Assay

(a) Quantitative Determination by Sandwich Assay of Collagenase Inhibitor in Sera from Normal Subjects and from Patients with Rheumatoid Arthritis

On the basis of the standard curve prepared in Example 7, fifty serum samples from normal subjects and fourteen from patients with rheumatoid arthritis were sandwich-assayed quantitatively for collagenase inhibitor contained therein. The results are shown in Table 5 below. In this sandwich assay there was used the human serum collagenase inhibitor purified in Example 6 as the antigen for preparation of the standard curve. This quantification was performed with the serum samples diluted 1,600 times with Buffer A containing 1% BSA. The values indicated in Table 5 are expressed as means of duplicate experiments. As is seen from Table 5, the mean amount of collagenase inhibitor present in one ml of serum from patients with rheumatoid arthritis was as high as 312±87 ng (p<<0.001), whereas that from normal subjects was 184±31 ng. Thus, it is possible, by quantitating collagenase inhibitor in serum by the sandwich assay, to know whether the subject is afflicted with rheumatoid arthritis.

(b) Quantitative Determination by Sandwich Assay of Collagenase Inhibitor in Plasmas from Normal Subjects and from Patients with Rheumatoid Arthritis

In accordance with the method of Ludlam and Cash as described in Br. J. Haematol. 33, 239-247 (1976), plasma was taken from bloods of normal subjects and of patients with rheumatoid arthritis. The plasma thus taken is the supernatant obtained from centrifugation at 1,900 xg for 60 min. at 4° C. of cooled blood supplemented with EDTA, prostaglandin E₁ and theophylline, wherein the platelets in the blood are retained in the precipitated fraction without decomposition.

The thus obtained twenty six plasma samples from normal subjects and twenty four from patients were sandwich-assayed quantitatively for collagenase inhibitor present therein by the same method as in (a) above. The results are shown in Table 6 below. As is seen from Table 6, the mean amount of collagenase inhibitor present in one ml of plasma from patients with rheumatoid arthritis was as high as 84±23 ng (p<<0.001), whereas that from normal subjects was 64±10 ng.

(c) Quantitative Determination by Sandwich Assay of Collagenase Inhibitor in Synovial Fluids from Normal Subjects and from Patients with Rheumatoid arthritis

Using the same method as shown in (a) above, seven synovial fluid samples from normal subjects and five from patients with rheumatoid arthritis were sandwich-assayed quantitatively for collagenase inhibitor contained therein. The results are shown in Table 7 below. As is seen from Table 7, the mean amount of collagenase inhibitor present in one ml of synovial fluid from patients with rheumatoid arthritis was as high as 1,424±26 ng (p<<0.001), whereas that from normal subjects was 357±51 ng.

Thus, it is possible, by quantitating collagenase inhibitor in synovial fluid, to know whether the subject is afflicted with rheumatoid arthritis.

EXAMPLE 9 Confirmation of Specific Reactivity Between Monoclonal Antibody and Collagenase Inhibitor

As was demonstrated in Example 4(b), the two monoclonal antibodies (clone 7-6C1 and clone 7-23G9) used in the sandwich assay exhibit reactivity with human collagenase inhibitor. A test was performed to establish that these monoclonal antibodies specifically react with collagenase inhibitor in such systems as sera, plasmas and synovial fluids in which different proteins are present dissolved in high concentrations. The sera from normal subjects and from patients with rheumatoid disease were diluted five times with a 30-mM Tris-HCl buffer solution (pH 8.0) containing 0.1-M NaCl and 5-mM CaCl₂, and then applied onto a clone 7-23G9 (antibody as the solid phase for the sandwich assay)-Sepharose 4B column to obtain the eluted fraction according to the manner as described in Example 4(b). After subjected to SDS-PAGE followed by Western blotting, the eluted fraction was immunologically stained with Fab'(clone 7-6C1)-POD conjugate, a labeled antibody for the sandwich assay. The results are shown in FIG. 7. As seen from FIG. 7, a single band was shown for all the cases (1: purified human serum collagenase inhibitor; 2: sera from normal subjects; and 3: sera from patients with rheumatoid disease). It has thus been demonstrated that according to the sandwich assay in the method of diagnosis of the invention collagenase inhibitor is determined extremely specifically.

As is apparent from the foregoing description, it is possible, with the method of the present invention, to perform the diagnosis of rheumatoid arthritis straightforwardly in a short period of time with high sensitivity.

                  TABLE 1                                                          ______________________________________                                         Clone No.          Subclass/Chain                                              ______________________________________                                         7-3F1              IgG1/κ                                                7-4F2              IgG1/κ                                                7-5A1              IgG1/κ                                                7-6C1              IgG1/κ                                                7-7F11             IgG1/κ                                                7-8B2              IgG2a/κ                                               7-9B4              IgG1/κ                                                7-10E11            IgG1/κ                                                7-11A5             IgG1/κ                                                7-12B6             IgG1/κ                                                7-14E9             IgG1/κ                                                7-15E8             IgG1/κ                                                7-18F3             IgG2b/κ                                               7-19F6             IgG1/κ                                                7-20C2             IgG1/κ                                                7-21B12            IgG1/κ                                                7-23G9             IgG1/κ                                                ______________________________________                                    

                  TABLE 2                                                          ______________________________________                                                 Fab'-POD Conjugate                                                     Solid Phase          IgG1      IgG1   IgG1                                     Antibody  IgG1 (7-3F1)                                                                              (7-6C1)   (7-19F6)                                                                              (7-21B12)                                ______________________________________                                         IgG1 (7-3F1)                                                                             0.011      3.989     2.252  2.684                                    IgG1 (7-4F2)                                                                             3.144      4.811     3.354  3.083                                    IgG1 (7-5A1)                                                                             3.257      0.010     0      0                                        IgG1 (7-6C1)                                                                             2.152      0.001     0.011  0                                        IgG1 (7-7F11)                                                                            0.125      0.239     0.102  0.067                                    IgG2a(7-8B2)                                                                             0          3.967     1.064  1.288                                    IgG1 (7-9B4)                                                                             0.196      0.057     0.029  0.020                                    IgG1 (7-10E11)                                                                           0.891      0.007     0.001  0.002                                    IgG1 (7-11A5)                                                                            3.406      4.455     3.288  3.260                                    IgG1 (7-12B6)                                                                            3.604      4.083     2.952  3.077                                    IgG1 (7-15E8)                                                                            0.313      0.781     0.387  1.897                                    IgG2b(7-18F3)                                                                            3.362      4.735     3.114  3.021                                    IgG1 (7-19F6)                                                                            1.066      0         0      0.004                                    IgG1 (7-20C2)                                                                            3.277      4.553     3.355  3.240                                    IgG1 (7-21B12)                                                                           2.107      0.002     0.003  0                                        IgG1 (7-23G9)                                                                            3.622      4.386     3.027  3.092                                    ______________________________________                                    

                  TABLE 3                                                          ______________________________________                                                               Sera                                                                           from patients with                                       Sera from normal subjects                                                                            rheumatoid arthritis                                     Sam- Collagenase                                                                               Sam-   Collagenase    Collagenase                              ple  inhibitor  ple    inhibitor                                                                               Sample                                                                               inhibitor                                No.  (μg/ml) No.    (μg/ml)                                                                              No.   (μg/ml)                               ______________________________________                                          1   0.82       26     1.03      1    1.33                                      2   0.87       27     1.50      2    1.42                                      3   1.40       28     1.23      3    2.02                                      4   1.07       29     1.63      4    2.02                                      5   1.17       30     1.33      5    1.74                                      6   1.40       31     1.40      6    2.85                                      7   1.33       32     1.40      7    2.02                                      8   1.27       33     1.17      8    3.54                                      9   1.03       34     1.10      9    2.28                                     10   1.40       35     1.17     10    1.96                                     11   1.03       36     1.17     11    2.53                                     12   1.33       37     1.23     12    1.90                                     13   1.13       38     1.63     13    1.71                                     14   1.40       39     1.03     14    1.74                                     15   0.82       40     1.40                                                    16   0.92       41     1.03                                                    17   1.03       42     1.33                                                    18   1.07       43     1.57                                                    19   1.17       44     1.53                                                    20   1.33       45     1.27                                                    21   1.47       46     1.10                                                    22   1.33       47     1.17                                                    23   1.13       48     1.40                                                    24   1.47       49     1.10                                                    25   1.00       50     1.03                                                    Mean 1.23 ± 0.20   Mean 2.08 ± 0.56                                      ______________________________________                                    

                  TABLE 4                                                          ______________________________________                                         Synovial fluids from   Synovial fluids from patients                           normal subjects        with rheumatoid arthritis                                        Collagenase           Collagenase                                     Sample   inhibitor     Sample  inhibitor                                       No.      (μg/ml)    No.     (μg/ml)                                      ______________________________________                                         1        2.30          1       8.04                                            2        2.04          2       12.33                                           3        2.56          3       7.78                                            4        2.30          4       10.76                                           5        3.02          5       8.54                                            6        2.04                                                                  7        2.39                                                                  Mean 2.38 ± 0.31 μg/ml                                                                          Mean 9.49 ± 1.77 μg/ml                            ______________________________________                                    

                  TABLE 5                                                          ______________________________________                                                               Sera                                                                           from patients with                                       Sera from normal subjects                                                                            rheumatoid arthritis                                     Sam- Collagenase                                                                               Sam-   Collagenase    Collagenase                              ple  inhibitor  ple    inhibitor                                                                               Sample                                                                               inhibitor                                No.  (ng/ml)    No.    (ng/ml)  No.   (ng/ml)                                  ______________________________________                                          1   123        26     155       1    200                                       2   131        27     225       2    213                                       3   210        28     185       3    303                                       4   161        29     245       4    303                                       5   176        30     200       5    261                                       6   210        31     210       6    428                                       7   200        32     210       7    303                                       8   191        33     176       8    531                                       9   155        34     165       9    342                                      10   210        35     176      10    294                                      11   155        36     176      11    380                                      12   200        37     185      12    285                                      13   170        38     245      13    257                                      14   210        39     155      14    261                                      15   123        40     210                                                     16   139        41     155                                                     17   155        42     200                                                     18   161        43     236                                                     19   176        44     230                                                     20   200        45     191                                                     21   221        46     165                                                     22   200        47     176                                                     23   170        48     210                                                     24   221        49     165                                                     25   150        50     155                                                     Mean 184 ± 31      Mean 312 ± 87                                         ______________________________________                                    

                                      TABLE 6                                      __________________________________________________________________________                           Plasmas from patients                                    Plasmas from normal subjects                                                                         with rheumatoid arthritis                                    Collagenase                                                                               Collagenase                                                                               Collagenase                                                                               Collagenase                               Sample                                                                             inhibitor                                                                             Sample                                                                             inhibitor                                                                             Sample                                                                             inhibitor                                                                             Sample                                                                             inhibitor                                 No. (ng/ml)                                                                               No. (ng/ml)                                                                               No. (ng/ml)                                                                               No. (ng/ml)                                   __________________________________________________________________________     1   70     15  51     1   60     15  49                                        2   75     16  57     2   74     16  61                                        3   84     17  53     3   110    17  119                                       4   70     18  60     4   65     18  82                                        5   54     19  60     5   79     19  109                                       6   49     20  64     7   88     20  88                                        7   74     21  56     7   91     21  119                                       8   73     22  62     8   89     22  95                                        9   64     23  74     9   59     23  125                                       10  72     24  61     10  65     24  115                                       11  62     25  78     11  81                                                   12  67     26  44     12  52                                                   13  54                13  68                                                   14  74                14  69                                                   Mean 64 ± 10       Mean 84 ± 23                                          __________________________________________________________________________

                  TABLE 7                                                          ______________________________________                                         Synovial fluids from   Synovial fluids from patients                           normal subjects        with rheumatoid arthritis                                        Collagenase           Collagenase                                     Sample   inhibitor     Sample  inhibitor                                       No.      (μg/ml)    No.     (μg/ml)                                      ______________________________________                                         1        345           1       1206                                            2        306           2       1850                                            3        384           3       1167                                            4        345           4       1614                                            5        453           5       1281                                            6        306                                                                   7        359                                                                   Mean 357 ± 51       Mean 1424 ± 296                                      ______________________________________                                    

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows Western blotting patterns of bovine dental pulp collagenase inhibitor subjected to SDS-PAGE, obtained with different monoclonal antibodies;

FIG. 2 shows a standard curve for bovine dental pulp collagenase inhibitor, prepared with a solid phase 7-23G9 antibody-conjugate Fab'(7-6C1)-POD assay system;

FIG. 3 shows immunologically stained patterns obtained by subjecting human serum collagenase inhibitor to SDS-PAGE and then to Western blotting;

FIG. 4 shows a standard curve for human serum collagenase inhibitor, prepared with a solid phase 7-23G9 antibody-conjugate Fab'(7-6C1)-POD assay system;

FIG. 5 shows SDS-PAGE patterns of collagenase inhibitor purified from human sera;

FIG. 6 shows a standard curve prepared by sandwich assay for purified human serum collagenase inhibitor; and

FIG. 7 shows Western blotting patterns obtained when the eluted fraction obtained upon application of human sera onto an affinity column with IgG(7-23G9) antibody bound was subjected to SDS-PAGE followed by application of Fab'(7-6C1)-POD conjugate. 

We claim:
 1. A method for diagnosing rheumatoid arthritis which comprises enzyme-immunologically measuring the amount of collagenase inhibitor present in sera, plasma or synovial fluid by means of a sandwich assay wherein two different monoclonal antibodies which specifically bind to different antigenic determinants of the collagenase inhibitor are used, wherein the first of said monoclonal antibodies is used for attachment of antigen to the solid phase and is an IgG-type antibody which is produced by a hybridoma having all of the identifying characteristics of the hybridoma deposited as BP-3469 and wherein the second of said monoclonal antibodies is enzyme conjugated and used to detect antigen bound by said first monoclonal antibody, said second antibody being produced by a hybridoma having all of the indentifying characteristics of the hybridoma deposited as BP-3468, and comparing the measured amount of collagenase inhibitor with that found in normal subjects. 